IL-6 Protein Expression Research Articles

Sevoflurane ameliorates LPS-induced inflammatory injury of HK-2 cells through Sirtuin1/NF-κB pathway.

The anesthetic sevoflurane (SEV) has been shown to protect against organ's injury during sepsis. The present study intended to uncover the protective effects of SEV on sepsis-induced acute kidney injury (SI-AKI) and its possible mechanism. Human renal tubular epithelial cell HK-2 was treated with 10 μg/mL lipopolysaccharide (LPS) to construct SI-AKI cell model. LPS-induced HK-2 cells were pretreated with SEV in the absence or presence of EX527, an inhibitor of Sirtuin 1 (SIRT1), after which were the detection of cell viability, lactate dehydrogenase (LDH) release, apoptosis, inflammation, and oxidative stress. Our results demonstrated that LPS caused decreased cell viability, increased LDH release, improved cell apoptosis along with decreased expression of Bcl2 and enhanced expressions of Bax, cleaved PARP and cleaved caspase, enhanced production, and protein expressions of TNF-α, IL-6, and IL-1β, increased generation of reactive oxygen species (ROS) and malondialdehyde (MDA), but contributed to declined activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). LPS inhibited SIRT1 and IκBα expressions but up-regulated p-NF-κB p65 and acetyl-p53 expressions as well. However, SEV pretreatment abolished all above-mentioned effects of LPS on HK-2 cells, while EX527 significantly reversed the effects of SEV. In conclusion, SEV effectively protected HK-2 cells against LPS-induced apoptosis, inflammation, and oxidative stress, and these effects may depend on the increase of SIRT1 expression, thereby inactivating NF-κB signaling.

18β-Glycyrrhetinic acid ameliorates endoplasmic reticulum stress-induced inflammation in pulmonary arterial hypertension through PERK/eIF2α/NF-κB signaling.

Endoplasmic reticulum stress (ERS)-induced inflammation participates in the occurrence of pulmonary arterial hypertension (PAH) by promoting pulmonary vascular remodeling, which involved in the activation of PERK/eIF2α/NF-κB signaling pathway. 18β-Glycyrrhetinic acid (18β-GA) has been found efficacious for attenuating PAH through its anti-remodeling effects in our previous research and it remains unclear whether 18β-GA has an effect on the remodeling caused by ERS-induced inflammation. In this study, we made observations in monocrotaline-induced PAH rats and found improvement of hemodynamic and histopathological parameters, decreases in the right ventricular hypertrophy index, and alleviation of pulmonary vascular remodeling after 18β-GA administration in vivo. Moreover, 18β-GA could significantly inhibit the proliferation and DNA synthesis of human pulmonary arterial smooth muscle cells (HPASMCs) induced by platelet-derived growth factor BB. At the cellular and molecular levels, we found that 18β-GA could significantly reduce the accumulation of misfolded protein in rat lung tissue, inhibit ERS activation, reduce the expression of GRP78, p-PERK, p-eIF2α, and p-NF-κB p65, and increase IκB protein expression. 18β-GA could inhibit the migration of NF-κB into the nucleus, reduce the contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatant of HPASMCs, and reduce GRP78, p-PERK, p-eIF2α, p-NF-κB p65, TNF-α, IL-6, and MCP-1 protein expression, increase IκB protein expression in HPASMCs. According to what we observed, this study indicated that 18β-GA could treat PAH, which is related to the inhibition of PERK/eIF2α/NF-κB signaling pathway.

Effect of Platycodon grandiflorus Polysaccharide on M1 Polarization Induced by Autophagy Degradation of SOCS1/2 Proteins in 3D4/21 Cells.

M1-polarized macrophages can improve the body’s immune function. This study aimed to explore the mechanism of Platycodon grandiflorus polysaccharide (PGPSt) degrading SOCS1/2 protein through autophagy and promoting M1 polarization in 3D4/21 cells. Immunoprecipitation, confocal laser scanning microscopy, flow cytometry, and intracellular co-localization were used to detect the expression of related phenotypic proteins and cytokines in M1-polarized cells. The results showed that PGPSt significantly promoted the mRNA expression of IL-6, IL-12, and TNF-α and enhanced the protein expression of IL-6, IL-12, TNF-α, IL-1β, iNOS, CD80, and CD86, indicating that PGPSt promoted M1 polarization in 3D4/21 cells. Next, the effect of the PGPSt autophagy degradation of SOCS1/2 on the M1 polarization of 3D4/21 cells was detected. The results showed that PGPSt significantly downregulated the expression level of SOCS1/2 protein, but had no obvious effect on the mRNA expression level of SOCS1/2, indicating that PGPSt degraded SOCS1/2 protein by activating the lysosome system. Further research found that under the action of 3-MA and BafA1, PGPSt upregulated LC3B II and downregulated SOCS1/2 protein expression, which increased the possibility of LC3B, the key component of autophagy, bridging this connection and degrading SOCS1/2. The interaction between SOCS1/2 and LC3 was identified by indirect immunofluorescence and Co-IP. The results showed that the co-localization percentage of the two proteins increased significantly after PGPSt treatment, and LC3 interacted with SOCS1 and SOCS2. This provides a theoretical basis for the application of PGPSt in the treatment or improvement of diseases related to macrophage polarization by regulating the autophagy level.

Chaihu Guizhi Decoction plus or minus formula combined with capecitabine inhibits IL-6/STAT3 signaling to suppress triple-negative breast cancer xenografts in nude mice

To investigate the effect of Chaihu Guizhi Decoction (CHGZD) combined with capecitabine on growth and apoptosis of subcutaneous triple-negative breast cancer xenografts in nude mice and explore the possible mechanism. Nude mouse models bearing subcutaneous triple-negative breast cancer xenografts were randomized into 6 groups (n=10) for treatment with distilled water (model group), low (10.62 g/kg), medium (21.23 g/kg) and high (42.46 g/kg) doses of CHGZD, capecitabine (0.2 mg/kg), or the combination of CHGZD (42.46 g/kg) and capecitabine (0.2 mg/k) once daily for 21 consecutive days. The general condition of mice was observed, and after 21-day treatments, the tumors were dissected for measurement of tumor volume and weight and histopathological examination with HE staining. Serum IL-6 levels of the mice were determined with enzyme-linked immunosorbent assay (ELISA), and the expression levels of IL-6, STAT3, p-STAT3, Bax, Bcl-2 and cyclin D1 in the tumor tissues were detected using real-time PCR and Western blotting. Compared with those in the model group, the tumor-bearing mice receiving treatments with CHGZD showed significantly increased food intake with good general condition, sensitive responses, increased body weight, and lower tumor mass (P < 0.01). Compared with capecitabine treatment alone, treatment with CHGZD alone at the medium and high doses and the combined treatment all resulted in significantly higher tumor inhibition rates (P < 0.01), induced obvious tumor tissue degeneration and reduced the tumor cell density. Treatments with CHGZD, both alone and in combination with capecitabine, significantly decreased serum IL-6 level, lowered the mRNA expression levels of IL-6 and STAT3, the protein expressions of IL-6, STAT3 and P-STAT3 (P < 0.05), and the mRNA and protein expressions of Bcl-2 and cyclin D1 (P < 0.05), and increased the mRNA and protein expressions of Bax in the tumor tissues (P < 0.05). CHGZD combined with capecitabine can significantly inhibit tumor growth in nude mice bearing triple-negative breast cancer xenografts, the mechanism of which may involve the inhibition of IL-6/STAT3 signaling pathway and regulation of Bax, Bcl-2 and cyclin D1 expressions to suppress tumor cell proliferation and differentiation and induce cell apoptosis.

D-aspartic acid protects against gingival fibroblasts inflammation by suppressing pyroptosis.

Peri-implantitis is the main cause of dental implant failure, which is associated with pyroptosis. The roles of D-aspartic acid (D-Asp) on pyroptosis and the mechanism of the protective effect of D-Asp on human gingival fibroblasts (HGFs) remain unknown. This study investigated the effects of D-Asp on the pyroptosis of HGFs induced by high mobility group box 1 protein (HMGB1). The cytotoxic effects of D-Asp on HGFs was detected by Cell Counting Kit-8 assay, the membrane permeability was investigated by propidium iodide/ Hoechst 33,342 double staining, flow cytometry analysis, and lactate dehydrogenase releasing, The gene and protein expression levels were detected by real-time quantitative PCR, enzyme-linked immunosorbent assay, and Western blot, respectively. Cell viability analysis showed that D-Asp ≤ 30 mM had no cytotoxicity to HGFs. HMGB1 drastically raised the membrane permeability of HGFs, while 1/10/30 mM D-Asp suppressed the permeability and remained the integrity of the membrane. HMGB1 promoted the mRNA expression of NLRP3, caspase-1, GSDMD, IL-1β, and IL-18, and the protein expression of IL-1β, IL-18, caspase-1, GSDMD, and NLRP3. With the pretreatment of HGFs with D-Asp of 1/10/30 mM for 24h, the cell membrane permeability was reduced and the expression of NLRP3, caspase-1, GSDMD, IL-1β, and IL-18 was significantly decreased compared with the HMGB1 group, indicating the competitive antagonism of D-Asp against HMGB1 on the binding with toll-like receptors. Hence, this study may provide a novel insight into preventing pyroptosis and propose a new strategy for the treatment of peri-implantitis.

Anti-inflammation effect of moxibustion for rats with diarrhea-predominant irritable bowel syndrome based on multiple miRNAs regulating NF-κB signal pathway

To observe the effect of moxibustion on the regulation of nuclear factor-kappa B (NF-κB) and inflammatory factors by multiple microRNAs (miRNAs) in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), and to explore the anti-inflammatory mechanism of moxibustion on IBS-D. Twelve of 52 newborn rats were randomly selected into a normal group. The remaining rats were made into IBS-D model. A total of 36 rats with successful model were randomly divided into a model group, a medication group and a moxibustion group, 12 rats in each group. The rats in the medication group were intraperitoneally injected with pyrrolidine dithiocarbamate (PDTC). The rats in the moxibustion group were treated with moxibustion at "Tianshu" (ST 25) and "Shangjuxu" (ST 37) for 20 min each time. All the intervention was given once a day for 7 days. Before and after modeling as well as after intervention, the body mass, loose stool rate and the minimum volume threshold of abdominal withdrawal reflex (AWR) were measured. After intervention, the contents of serum tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-8 were detected by ELISA method; the morphology of colon tissues was observed by HE staining, and the expressions of miR-155, miR-125b, miR-29b, miR-31, miR-18a and NF-κB p65 mRNA in colon tissues were detected by real-time PCR. The expressions of NF-κB p65, TNF-α, IL-1β and IL-8 protein in colon tissues were detected by immunofluorescence. After modeling, the body mass and the minimum volume threshold of AWR in the model group were lower than those in the normal group (P<0.01); the rates of loose stool in the model group were higher than those in the normal group (P<0.01); after intervention, in the model group, the inflammatory infiltration of colon tissues was obvious, and the serum levels of TNF-α, IL-1 β, IL-8 were higher than those in the normal group (P<0.05); the expression of miR-155, miR-125b, miR-29b, miR-31, miR-18a and NF-κB p65 mRNA in colon tissues was higher than that in the normal group (P<0.05); the protein expression of NF-κB p65, TNF-α, IL-1β, IL-8 was also higher than that in the normal group (P<0.01). After intervention, the body mass and the minimum volume threshold of AWR in the medication group and the moxibustion group were both higher than those in the model group (P<0.05); the loose stool rate in the medication group and the moxibustion group were lower than those in model group (P<0.05); the inflammatory cells infiltration in the colon tissues was less, the serum levels of TNF-α, IL-1β and IL-8 as well as the protein expression of NF-κB p65, TNF-α, IL-1β and IL-8 in the colon tissues in the medication group and the moxibustion group were lower than those in the model group (P<0.05, P<0.01). The expression of miR-125b, miR-31, miR-18a and NF-κB p65 mRNA in the medication group were lower than those in the model group (P<0.05). The expression of miR-155, miR-125b, miR-29b, miR-31, miR-18a and NF-κB p65 mRNA in the moxibustion group were lower than those in the model group (P<0.05). The miR-155, miR-125b, miR-29b, miR-31, miR-18a were positively correlated with NF-κB p65 mRNA (0<r<1, P<0.01). The anti-inflammatory mechanism of moxibustion at "Tianshu" (ST 25) and "Shangjuxu" (ST 37) for IBS-D rats may be related to regulating multiple miRNAs to inhibit NF-κB signal pathway and reduce the expression of inflammatory factors.

Effects of Long-Term Exposure to PM2.5 on Oxidative Stress Injury and Expression of Inflammatory Factors, NF-κB p65 and Cx43 in Bone Marrow of Mice

Objective: This study aims to explore the toxic effect of PM2.5 on the hematopoietic microenvironment of the bone marrow, and investigate the effect of PM2.5 on oxidative stress injury, the secretion of inflammatory cytokines and the expression of NF-κB p65 and C x 43 in the bone marrow of mice.Methods: A total of 615 mice were treated with normal saline, low dose (0.1 mg/ml) PM2.5 suspension, and high dose (0.2 mg/ml) PM2.5 suspension by intratracheal instillation, respectively. The ROS content, activity of total SOD (T-SOD), DNA injury, and the protein expression levels of IL-1 β, IL-6, TNF-α, NF-κB p65 and Cx43 in bone marrow cells (BMCs) of mice were detected in these three groups.Results: Compared with the normal saline control group, the expression level of reactive oxygen species (ROS) significantly increased in BMCs, while the activity of SOD enzymes significantly decreased in PM2.5 exposed mice. Furthermore, DNA injury significantly increased in PM2.5 exposed mice, and the expression of IL-1 β, IL-6, TNF-α and NF-κB p65 significantly increased, while the protein expression of Cx43 significantly decreased in the PM2.5 exposed mice. The differences were statistically significant (p &amp;lt; 0.05). There were significant differences in ROS content, T-SOD activity, DNA injury (TL, TD and TM), protein expression of IL-1β, IL-6, TNF-α and Cx43 between the high-dose PM2.5 exposure group and low-dose PM2.5 exposure group (p &amp;lt; 0.05), but there was no significant difference in the expression of NF-κB p65 between the high-dose and low-dose PM2.5 exposure groups (p &amp;gt; 0.05).Conclusion: PM2.5 has toxic effects on the bone marrow of mice. Oxidative stress injury, inflammatory reaction and abnormal intercellular communication may be the underlying mechanism.

Network pharmacology approach and molecular docking to explore the potential mechanism of Wu-Wei-Wen-Tong Chubi capsules in rheumatoid arthritis.

Network pharmacology, a holistic approach based on the theory of biological network technology, integrates information from biological systems, drugs, and diseases. Here, this theory was used to predict the targets of Wu-Wei-Wen-Tong Chubi capsule (WWWT) to explore the mechanism in the treatment of rheumatoid arthritis (RA). The ingredients of each herbal medicine in WWWT were collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the active ingredients were screened through bioavailability (OB) ≥30% and drug-likeness (DL) ≥ 0.18. SwissTargetPrection and TCMSP were utilized to calculate and predict the targets of active ingredients. RA-related targets were obtained by searching the Genecards and OMIM databases. The common targets of RA and WWWT were used for gene ontology (GO), KEGG pathway enrichment, protein-protein interaction (PPI) analysis, and molecular docking. And then, four key genes were screened for subsequent verification experiments. In total, 90 active compounds and 330 potential targets of WWWT, 1310 targets of RA, and 135 intersection targets were found. Additionally, GO and pathway analysis identified 4610 significant GO terms and 147 significant KEGG pathways. Based on the PPI network, 11 key genes including IL-6, MMP-9, and TNF-α were screened out for molecular docking. Molecular docking showed that these key genes have good binding activities to active compounds of WWWT such as oroxylin a, kaempferol, and luteolin. Simultaneously, Western blot experimental validation demonstrated that the protein expressions of IL-6, MMP-9, TNF-α, and VEGFA significantly decreased after WWWT treatment. The mechanism of WWWT in treating RA involves multiple active compounds acting on multiple targets, and multiple pathways, which provides an important reference for further elucidation the mechanism and clinical applications of WWWT in the treatment of RA.

Improved oral nutraceutical-based intervention for themanagement of obesity: pterostilbene-loaded chitosan nanoparticles.

Aim: To formulate and assess the oral anti-obesity effect of polymeric-based pterostilbene (PS)-loaded nanoparticles. Methods: Pterostilbene-hydroxypropyl β-cyclodextrin inclusion complex loaded in chitosan nanoparticles (PS/HPβCD-NPs) were prepared and characterized in vitro. Cytotoxicity, pharmacokinetics and anti-obesity effects were assessed on Caco-2 cell line and high-fat-diet-induced obesity rat model, respectively. In vivo assessment included histological examination, protein and gene expression of obesity biomarkers in adipose tissues. Results: Safe PS/HPβCD-NPs were successfully prepared with improved bioavailability compared with free PS. PS/HPβCD-NPs showed an improved anti-obesity effect, as supported by histological examination, lipid profile, UCP1 gene expression and protein expression of SIRT1, COX2, IL-6 and leptin. Conclusion: Orally administered PS nanoparticles represent a new and promising anti-obesity strategy owing to the sustainable weight loss and minimal side effects; this may be of great socio-economic impact.

GPR120 modulates epileptic seizure and neuroinflammation mediated by NLRP3 inflammasome

BackgroundThe complex pathophysiology of epilepsy hampers the development of effective treatments. Although more than ten kinds of anti-seizures drugs (ASDs) have good effects on seizure control worldwide, about 30% of patients still display pharmacoresistance against ASDs. Neuroinflammation seems to play a crucial role in disease progression. G protein-coupled receptor 120 (GPR120) has been shown to negatively regulate inflammation and apoptosis. However, the role of GPR120 in epilepsy remains unclear. In this study, we aimed to explore the mechanism of GPR120 in epilepsy.MethodsMale adult C57BL/6 mice were intracranially injected with kainic acid (KA) to establish epilepsy model, and the adeno associated virus (AAV) was administered intracranially at 3 weeks before KA injection. VX765 was administered by intragastric administration at 30 min before KA induced and an equal dose administrated twice a day (10 a.m. and 4 p.m.) lasting 7 days until the mice were killed. Western blot analysis, immunofluorescence staining, video monitoring of seizure, LFP recording, Nissl staining were performed.ResultsGPR120 was increased in both the hippocampus and cortex in the KA-induced model with temporal lobe epilepsy (TLE), and both were most highly expressed at 7 days after KA injection. Overexpression of GPR120 significantly alleviated epileptic activity, reduced neuronal death after status epilepticus (SE), downregulated the expression of IL-1β, IL-6, IL-18, and pyrin domain-containing protein 3 (NLRP3) inflammasome, whereas knockdown GPR120 showed the opposite effect. The effects of GPR120 knockdown were reversed by VX765 inhibition cysteinyl aspartate specific proteinase-1 (Caspase-1).ConclusionGPR120 modulates epileptic seizure activity and affects neuronal survival in KA-induced mouse model of temporal lobe epilepsy. Furthermore, GPR120 regulated neuroinflammation in epileptic animals through NLRP3/Caspase-1/IL-1β signaling pathway.

Salvia miltiorrhiza polysaccharides ameliorates Staphylococcus aureus-induced mastitis in rats by inhibiting activation of the NF-κB and MAPK signaling pathways

The lactation capacity of dairy cows is critical to the productivity of the animals. Mastitis is a disease that directly affects the lactation capacity of cows. Staphylococcus aureus (S. aureus) is one of the most important pathogens that causes mastitis in dairy cows. The anti-inflammatory effect of Salvia miltiorrhiza polysaccharides (SMPs) has been demonstrated in mice and chickens. However, the effectiveness of SMPs in preventing and treating mastitis is unclear. Therefore, the purpose of this study was to explore the protective effect and mechanism of SMPs on mastitis caused by S. aureus. S. aureus was used to induce mastitis in rats, and three doses of SMPs (87.5, 175, 350 mg/kg, BW/d) were administered as treatments. The bacterial load, histopathology, and myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAGase) activities of mammary glands were observed and measured. Cytokines, including interleukin (IL)-1β, interleukin (IL)-6, and tumor necrosis factor α (TNF-α), were examined by qRT–PCR and ELISA. Key proteins in the NF-κB and MAPK signaling pathways were analyzed by Western blotting. The results showed that SMP supplementation could significantly reduce the colonization of S. aureus and the recruitment of inflammatory cells in mammary glands. S. aureus-induced gene transcription and protein expression of IL-1β, IL-6, and TNF-α were significantly suppressed in mammary glands. In addition, the increase in NF-κB and MAPK protein phosphorylation was inhibited by SMPs. These results revealed that supplementation with SMPs protected the mammary gland of rats against damage caused by S. aureus and alleviated the inflammatory response. This study provides a certain experimental basis for the treatment of S. aureus-induced mastitis with SMPs in the future.

Mechanism of electroacupuncture against intestinal inflammation in mice with Parkinson's di-sease by NF-κB/IL-6 pathway

To observe the effects of electroacupuncture (EA) at "Fengfu"(GV16), "Taichong"(LR3), and "Zusanli"(ST36) on the tyrosine hydroxylase (TH), nuclear factor-kappa B (NF-κB), interleukin (IL)-6, and zonula occludens-1 (ZO-1) in mice with Parkinson's disease (PD), and to explore the underlying mechanism of EA in the treatment of PD. C57BL/6 mice were randomly divided into control, model, and EA groups, with 10 mice in each group. The PD model was induced by rotenone (i.g.) in mice for 28 d. EA was applied to GV16, LR3 and ST36 of mice in the EA group for 30 min, once daily for 14 d. The behavioral changes of mice in each group before and after treatment were observed and scored. The total distance traveled autonomously of mice was detected in the open field test. TH expression in the substantia nigra (SN) was measured by immunohistochemistry, and the changes in colon tissue structure were observed by HE staining. The intestinal mRNA expression of ZO-1, NF-κB, and IL-6 was detected by quantitative real-time PCR, and the colon tissue protein expression of NF-κB was detected by Western blot. The colon tissue concentration of IL-6 was detected by ELISA. Compared with the control group, the pre- and post-treatment behavioral scores of the model group and the pre-treatment behavioral score of the EA group were increased (P<0.01). Compared with the model group, the EA group showed reduced behavioral score after treatment (P<0.01). Compared with the control group, the model group showed reduced goblet cells and crypts and thinner muscle layer in the intestinal specimens of mice. Compared with the model group, the EA group showed intact surface villi, increased goblet cells and crypts, and thickened muscle layer. Compared with the control group, the model group exhibited reduced total distance traveled in the open field test, TH expression in SN, and ZO-1 mRNA expression (P<0.01), and elevated mRNA and protein expression of NF-κB and IL-6, and the ratio of p-NF-κB/NF-κB in the intestinal tract (P<0.01). Compared with the model group, the EA group displayed increased total distance traveled, TH expression in SN, and ZO-1 mRNA expression (P<0.01), and declining mRNA and protein expression of NF-κB and IL-6, and the ratio of p-NF-κB/NF-κB in the colon tissue (P<0.01, P<0.05). EA at GV16, LR3, and ST36 can regulate the expression of NF-κB/IL-6, inhibit the transmission of the colon tissue inflammatory response, repair the intestinal barrier function, and potentiate the TH activity, thereby improving the behavioral performance of PD mice.

Accelerated wound healing and its promoting effects of topical codeine on the healing of full-thickness cutaneous wound, evidences for modulating cytokines involved in pain, inflammation and collagen biosynthesis.

The inflammation and pain occur in all the wounds. Opioids drugs decrease pain and may act as an anti-inflammation. The current study was conducted to investigate the efficiency of the topical uses of Codeine on full-thickness excision wound models by focusing on relationship between pain mediators, inflammation and wound healing rate. Following the induction of anesthesia, a skin wound with a size of 7-mm punch was induced on the dorsal surfaces of each mouse. The mice were divided into five categories: groups I-III were daily administered 2.5%, 5%, and 10% Codeine gel; those in group IV were administered phenytoin cream, and group V (controls) received base ointment. To assess the effects of Codeine gel on the wound healing process, the wound area, histological parameters, and the relative protein expression of CXCR1, CXCR2, IL-6, IL-6R, PDGF, PDGFR, and COL1A along with the plasma concentrations of IL-1β, IL-10, and TNF-α were investigated on days 3, 7, and 14. On days 7 and 14, the wound area was significantly lower in the treated mice compared to the controls (P < 0.05). Angiogenesis, collagen deposition, and epithelium thickness were significantly higher in the treatment groups compared to the control group (P < 0.05). The relative protein expressions of CXCR1, CXCR2, IL-6, and IL-6R and the plasma concentrations of IL-1β and TNF-α were significantly lower in the treated groups. Meanwhile, the relative protein expressions of PDGF, PDGFR, and COL1A and the plasma concentration of IL-10 were significantly higher in the treated mice (P < 0.05). Administration of Codeine gel accelerated wound healing through decreasing the pain mediators, inflammation and promoting proliferative phase.

The IL-6/STAT3 signaling pathway mediated by radiotherapy regulates the expression of PD-L1 in esophageal cancer cells

Objective: To explore the possible mechanism of radiotherapy regulating the expression of PD-L1 in esophageal carcinoma. Methods: Three esophageal cancer cell lines (Eca109, Kyse150, TE1) were irradiated with different doses of X-rays, and 6 Gy+ AG490 group was set. The mRNA expression of PD-L1 was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The protein expressions of PD-L1, STAT3, p-STAT3 were detected by western blotting and the protein level of IL-6 was detected by ELISA. Results: The mRNA expressions of PD-L1 in Eca109, Kyse150 and TE1 were 2.86±0.30, 960.01±21.27 and 106.78±6.67, higher than 1.07±0.15 in normal esophageal cell line HET-1A (P<0.01). The protein expressions of PD-L1 in Eca109, Kyse150 and TE1 were 0.091±0.036, 1.533±0.079 and 0.914±0.035, higher than 0.063±0.01 in normal esophageal cell line HET-1A (P<0.01). After 48 hours of 6 Gy irradiation, the protein expression levels of PD-L1 in Eca109, Kyse150 and TE1 were 0.135±0.007, 1.66±0.06 and 1.32±0.06, higher than 0.09±0.01, 1.21±0.05 and 0.93±0.03 of the 0 Gy group (P<0.01), while the protein expression levels of p-STAT3 in Eca109, Kyse150 and TE1 were 1.44±0.26, 0.75±0.04 and 1.92±0.17, higher than 0.18±0.05, 0.48±0.02 and 0.36±0.06 of the 0 Gy group (P<0.01). IL-6 protein expression increased significantly after different doses of irradiation (P<0.01). After the IL-6/STAT3 signaling pathway was blocked by the specific inhibitor AG490, the expressions of PD-L1 of Eca109, Kyse150 and TE1 in the 6 Gy+ AG490 groups were 0.11±0.03, 1.07±0.08 and 0.96±0.11, without significant differences of 0.09±0.01, 0.96±0.05 and 0.85±0.09 of the 0 Gy group (P>0.05), while the protein expressions of p-STAT3 were 0.76±0.11, 0.59±0.06 and 0.96±0.12, without significant differences of 0.67±0.08, 0.54±0.06 and 0.84±0.11 of the 0 Gy group (P>0.05). Conclusion: Radiotherapy may regulate the expression of PD-L1 in esophageal cancer cells through IL-6 / STAT3 signaling pathway.

Delay of Feed Post-Hatch Causes Changes in Expression of Immune-Related Genes and Their Correlation with Components of Gut Microbiota, but Does Not Affect Protein Expression

Simple SummaryNewly hatched chicks do not have access to feed until between 48 and 72 h post-hatch based on standard practices in the poultry industry. How these practices affect the chicken’s immune system in not well understood. In this study, we investigated the effect of a delay in access to feed for 48 h in newly hatched chicks on the expression of various immune-related genes in the ileum and analyzed the correlation between these genes and the components of the ileal microbiota. The results suggest that several immune-related genes were affected by delayed access to feed and the age of the birds; however, these changes were transient, occurring mostly within 48 h of the return of birds to feed. In the correlation analysis between gene expression and components of the ileal microbiota, an increased number of significant correlations between immune-related genes and the genera Clostridium, Enterococcus, and the species Clostridium perfringens suggests a perturbation of the immune response and ileal microbiota in response to lack of feed immediately post-hatch. These results point out the complexity of the interplay between microbiota and the immune response and will help further explain the negative effects of delay in access to feed on production parameters in chickens.Because the delay of feed post-hatch (PH) has been associated with negative growth parameters, the aim of the current study was to determine the effect of delayed access to feed in broiler chicks on the expression of immune-related genes and select proteins. In addition, an analysis of the correlation between gene expression and components of the gut microbiota was carried out. Ross 708 eggs were incubated and hatched, and hatchlings were divided into FED and NONFED groups. The NONFED birds did not have access to feed until 48 h PH, while FED birds were given feed immediately PH. The ileum from both groups (n = 6 per group) was sampled at embryonic day 19 (e19) and day 0 (wet chicks), and 4, 24, 48, 72, 96, 144, 192, 240, 288, and 336 h PH. Quantitative PCR (qPCR) was carried out to measure the expression of avian interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-18, transforming growth factor (TGF-β), toll-like receptor (TLR)2, TLR4, interferon (IFN)-β, IFN-γ, and avian β-defensins (AvBD) I, 2, 3, 5, 6, 7, 8, 9, and 10. Protein expression of IL-10, IL-1β, IL-8, and IL-18 were measured using ELISAs. A correlation analysis was carried out to determine whether any significant association existed between immune gene expression and components of the ileal luminal and mucosal microbiota. Expression of several immune-related genes (TGF-β, TLR4, IFN-γ, IL-1β, IL-4, IL-6, and AvBDs 8 and 9) were significantly affected by the interaction between feed status and age. The effects were transient and occurred between 48 and 96 h PH. The rest of the genes and four proteins were significantly affected by age, with a decrease in expression noted over time. Correlation analysis indicated that stronger correlations exist among gene expression and microbiota in NONFED birds. The data presented here indicates that delay in feed PH can affect genes encoding components of the immune system. Additionally, the correlation analysis between immune gene expression and microbiota components indicates that a delay in feed has a significant effect on the interaction between the immune system and the microbiota.

High-Temperature Soup Foods in Plastic Packaging Are Associated with Phthalate Body Burden and Expression of Inflammatory mRNAs: A Dietary Intervention Study.

Plastic packaging material is widely used to package high-temperature soup food in China, but this combination might lead to increased exposure to phthalates. The health effects and potential biological mechanisms have not been well studied. This study aimed to examine urinary phthalate metabolites and the expression of inflammatory cytokines in the blood before, during, and after a "plastic-packaged high-temperature soup food" dietary intervention in healthy adults. The results showed that compared with those in the preintervention period, urinary creatinine-adjusted levels of monomethyl phthalate (MMP), mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MIBP), and total phthalate metabolites in the intervention period were significantly higher, with increases of 71.6, 41.8, 38.8, and 29.8% for MMP, MBP, MIBP, and the total phthalate metabolites, respectively. After intervention, the mean levels of IL-1β, IL-4, and TNF-α mRNA increased by 19.0, 21.5, and 25.0%, respectively, while IL-6 and IFN-γ mRNA decreased by 24.2 and 32.9%, respectively, when compared with the preintervention period. We also observed that several phthalates were associated with the mRNA or protein expression of IL-8, TNF-α, and IL-10. Therefore, consumption of plastic-packaged high-temperature soup food was linked to increased phthalate exposure and might result in significant changes in mRNA expression of several inflammatory cytokines.

Interleukin 6/gp130 axis promotes neural invasion in pancreatic cancer.

Nerve invasion (N-inv) is an important prognostic factor in pancreatic ductal adenocarcinoma (PDAC). Elucidation of circulating N-inv stimulators could provide deeper insights and novel perspectives for PDAC therapy. The interleukin (IL)-6/gp130 axis was evaluated in this study as a candidate N-inv stimulator. A human pancreatic cancer (PC) cell, Capan-1, was confirmed to have the stimulant activity of IL-6/gp130 axis through the evaluation of mRNA, cell surface protein and intracellular protein levels and chemotaxis and wound healing assay. The upregulation of IL-6/gp130 axis was evaluated using tumor-derived IL-6 level and intratumoral pSTAT3 expression in N-inv of murine sciatic nerves by intraneural injection of Capan-1 cell (N-inv model) and using resected pancreatic cancer tissue and clinical data from 46 PDAC patients. mRNA and protein expressions of IL-6 and IL-6 receptor were found in whole cell lysate and condition medium from PC cell. Cell surface protein expression of gp130 were clearly detected on PC cell. IL-6 promoted migration and chemotaxis of PC cell. Serum IL-6 and tumoral IL-6 mRNA levels in N-inv model mice were significantly higher than those in subcutaneous tumor mice (p = 0.004 and p = 0.002, respectively). Silencing of IL-6 and gp130 on PC cell and administration of an anti-IL-6 receptor antibody, tocilizumab, suppressed N-inv, compared to each control (p = 0.070, p = 0.118 and p = 0.122, respectively). In PDAC patients, the high-N-inv group showed poor prognosis (p =0.059) and elevated serum levels of IL-6 and C-reactive protein, synthesis of which is promoted by IL-6, compared to those in the low-N-inv group (p = 0.006 and p = 0.075, respectively). Tumoral gp130 expression at N-inv was higher than that in the primary pancreatic tumor (p = 0.026). Biological activity of IL-6/gp130 axis promoted N-inv in murine model and was upregulated in PDAC patients with severe N-inv. This study is the first evidence that the IL-6/gp130 axis offers a potential therapeutic target in PDAC with N-inv.

Trypanosoma cruzi dysregulates expression of piRNA that can regulate IL‐6 signaling in human cardiac fibroblasts during the early phase of infection

Trypanosoma cruzi, the etiological agent of Chagas Disease, causes severe morbidity, mortality, and economic burden in endemic zones. Due to globalization, the disease is now present in all industrialized regions of the world. About 40% of infected individuals will develop cardiovascular, neurological, and/or gastrointestinal pathologies. Cardiomyopathies induced by chronic parasite infection include organ enlargement and fibrosis, accompanied by significant changes in the cellular expression of profibrotic factors and inflammatory molecules. Accumulating evidence suggests that the parasite induces alterations in host gene expression profiles to facilitate infection and pathogenesis. The role of regulatory gene expression machinery during T. cruzi infection remains to be elucidated. In this study, we aim to evaluate the expression of a class of small non‐coding RNA molecules, piwi‐interacting RNAs (piRNAs), during the early phase of T. cruzi infection in primary human cardiac fibroblasts (PHCF). Recent studies defined a role for STAT3 signaling in the onset of cardiac fibrosis in a Chagasic mouse model. Our bioinformatic analysis of parasite challenged PHCF identified significant modulation of piRNAs targeting IL‐6 signaling pathway components. To validate this, we performed qPCR of IL‐6, SOCS3, and piRNAs predicted to target IL‐6 and SOCS3. Furthermore, we evaluated protein expression for pSTAT3 (Y705) and SOCS3 in cell lysates and IL6 secretion in condition media of parasite challenged PHCF. We observed that the piRNAs expression negatively correlated with their target mRNA and protein expression of IL‐6. This suggests T. cruzi dysregulates the expression of piRNA that can regulate IL‐6 signaling during T. cruzi pathogenesis.

Brain Kinin B1 Receptor Antagonism Attenuates Angiotensin II‐Induced Neurogenic Hypertension

Hypertension remains the leading cause of death globally, accounting for 10.4 million deaths per year. Recent evidence has shown that the kallikrein‐kinin system may play a vital role in the neural control of hypertension, through interactions with key regulatory systems such as the renin‐angiotensin system (RAS). We previously showed that kinin B1 receptor (B1R) activation promotes the development of DOCA‐salt‐induced neurogenic hypertension. However, accumulated evidence has shown that elevated levels of angiotensin II (Ang II) in the brain or upregulated neuronal angiotensin II type I receptor (AT1R) expression, results in increased neuroinflammation and the development of neurogenic hypertension. Recently, we presented data supporting B1R blockade prevents Ang II induced inflammation and oxidative stress in primary hypothalamic neurons. Yet, the interactions between the B1R and AT1R have not been fully studied. In this study, we hypothesize that Ang II treatment will increase blood pressure through B1R mediated mechanisms. Ang II infusion (600 ng/kg/min, SC, 2 weeks) increased blood pressure (radiotelemetry) in wild‐type (WT) male mice (145 ±13 mmHg vs. 103 ±6, n=6, p&lt;0.01). This increase in blood pressure was attenuated in B1R knockout (B1RKO) mice with Ang II infusion (118 ±12 mmHg, n=6, p&lt;0.01). Interestingly, our data showed no significant differences in B1RKO male and female mice with Ang II‐infusion. Additionally, B1RKO mice show reduced Ang II‐induced AT1R expression in the brain. Using ultra‐sensitive mass spectrometry detection, we found that Ang II‐induced an increase in B1R specific agonist Lys‐Des‐Arg9‐Bradykinin (LDABK) within the hypothalamus in WT mice (p&lt;0.01, n=5). More specifically, immunofluorescence staining showed that B1R is upregulated within neurons in the paraventricular nucleus (PVN) of the hypothalamus, a central regulatory center for cardiovascular and autonomic control. Ang II‐treated mice had increased inflammation (qRT‐PCR), as shown by elevated gene and protein expression of TNFα, IL‐1β, and IL‐6 (6‐, 5‐ and 8‐fold respectively, n=5, p&lt;0.01 vs. control) in the PVN, which was attenuated in B1RKO mice. To further determine interactions between AT1R and B1R, we used proximity ligation assay (PLA), to elucidate close proximity (&lt;40 nM) receptor‐receptor interactions indicated by fluorescent PLA signal. Our results showed that AT1R‐B1R close proximity interactions are detected within the PVN, and the PLA signals are significantly increased by Ang II treatment in WT mice (p&lt;0.01, n=5). Furthermore, to supplement our findings we isolated cultured primary hypothalamic neurons from WT and B1RKO mice and investigated the interaction between Ang II and B1R activation. Primary WT neurons treated with Ang II (300 nM, 24 hr) showed increased B1R expression, inflammation, and AT1R‐B1R interaction. However, B1RKO neurons or WT neurons pretreated with R715 (a B1R specific peptide antagonist) showed an attenuation of these effects. These results indicate B1R blockade attenuates Ang II‐induced hypertension through novel B1R and AT1R interactions. Collectively, our data provide distinct evidence suggesting B1R blockade could serve as a therapeutic target in the treatment of hypertension.

Liraglutide Is Protective against Brain Injury in Mice with Febrile Seizures by Inhibiting Inflammatory Factors.

The febrile seizure (FS) is a common disease in emergency pediatrics, and about 30% of patients are children aged between 6 months and 5 years. Therefore, we aim to observe the protective impact of liraglutide (LIR) on brain injury in mice with FS and to explore its relevant mechanisms. Male SD mice were selected, and the FS model was established by heat bath method. The behavioral score was performed on mice with Racine grading, and nerve cells in apoptosis in the hippocampus were determined by TUNEL. The content of glutamate was determined by ELISA. mRNA levels and protein expression of GLP-1, GLP-1R, IL-1β, IL-6, TNF-α, and cleaved-caspase 3 were examined in mice by q-PCR and WB. Protein expression of γ-aminobutyric acid was influenced by WB as well. LIR prolonged the seizure latency and seizure duration in mice with FS. The GLP-1 and GLP-1R in the mouse hippocampus with FS expressed highly and also inhibited the number of nerve cells in apoptosis, decreased glutamate content, and increased γ-aminobutyric acid expression in the mouse hippocampus with FS. In addition, The IL-1β, IL-6, and TNF-α, in the mouse hippocampus with FS expressed to reduce with LIR. LIR is protective against brain injury in mice with FS and protects brain injury by inhibiting inflammatory factors in mice with FS. Our finding provides a reference for mitigating and delaying the development of FS as well as the prevention and treatment of brain injury caused by FS.